Cryo-EM Reveals Two Distinct Serotonin-Bound Conformations of Full-Length 5-HT3A Receptor

Abstract

validate structural predictions using solution-phase small angle neutron scattering (SANS). We have acquired SANS data from GLIC under nonactivating buffer conditions, hiding the detergent micelle by use of deuteration. Using structures and molecular dynamics simulations of different states, the agreement with the SANS data has been evaluated. Although differences in the fit of the predicted models to the experimental data were small, our work shows that a resting state model is consistent with the room-temperature experimental data. This work shows the potential for using SANS in conjunction with other methods in the study of membrane proteins, while underlining the importance of high quality SANS data of multiple states. . Serotonin receptors (5-HT 3A R) are cation-selective, neurotransmitter-gated, pentameric ion channels that play a crucial role in regulating cellular excitability. 5-HT 3A R activities directly regulate gut movement and therefore drugs that inhibit 5-HT 3A R function are used to control vomiting reflexes. Serotonin binding induces a global conformational change encompassing the ligandbinding extracellular domain (ECD), the transmembrane domain (TMD), and the intracellular domain (ICD), the molecular details of which are unclear. Here, we determined two serotonin-bound structures of the full-length 5-HT 3A R in distinct conformations at 3.32 Å and 3.89 Å resolutions, which we referred as State 1 and State 2, respectively. The new structures reveal the mechanism underlying channel activation. When compared to Apo-5-HT 3A R, serotonin-bound states underwent a large twisting motion in the ECD and TMD leading to the opening of a 165 Å long permeation pathway. Notably, this motion results in creation of lateral portals for ion permeation at the interface of the TMD and ICD. Combined with molecular dynamics simulations, these structures provide novel insights into conformational coupling across domains and functional modulation.

A Heuristic Derived from Analysis of the Ion Channel Structural Proteome Permits the Rapid Identification of Hydrophobic Gates

“…The concept of hydrophobic gating in members of the Cys-loop family of ligand-gated ion channels, including the nicotinic acetylcholine receptor (10), GLIC (7,11), and the 5-HT3 receptor (12)(13)(14) is now well established. However, experimental and computational evidence of hydrophobic gates and barriers within other ion channels has also emerged, including for the TWIK-1 K2P channel (15), BK channels (16), the CorA magnesium channel (17), the CRAC biorxiv_global_text_v28_figs_SI.docx channel Orai (18), and members of the transient receptor potential (TRP) channel family (19)(20)(21)(22).…”

A Refined Open State of the Glycine Receptor Obtained Via Molecular Dynamics Simulations

“…Simulations of open-like states of the pLGIC superfamily undergo a hydrophobic collapse once artificial restraints on the protein structure are released 29-32 . This is why it has become common practice to apply restraints on the protein backbone to artificially fix its position 24,28,37 . Recently, 38 reported that while simulations starting from one structure of the 5-HT 3 receptor (I2, PDB: 6HIQ) gave the familiar hydrophobic collapse, simulations starting from another (F, PDB: 6HIN) allowed ions and water to flow throughout the trajectory and that wetting was correlated to rotation of L9' out of the pore lumen.…”

Electric Field Induced Wetting of a Hydrophobic Gate in a Model Nanopore Based on the 5-HT3Receptor Channel

“…To quantify the effects of electrowetting of a hydrophobic gate, we have employed MD simulations of the M2 helix nanopore which corresponds to the pore lining segment of the 5-HT3 receptor channel 16 . This model nanopore forms an ideal system for studying hydrophobic gating because it embodies many aspects of the wetting/de-wetting behaviour observed in complex ion channel proteins 38 , whilst remaining sufficiently simple to allow functional dissection by an extended series of atomistically detailed simulations ( Fig.…”

“…The gating or regulation of hydrophobic gates in ion channels is thought to involve either structural changes in pore radii, or more subtle changes in hydrophobicity through e.g. rotation of helices that contain side chains of different polarity and examples of such hydrophobic gating mechanism have now been proposed in a variety of different channels 12,[14][15][16][17][18][19][20][21][22][23][24][25] and synthetic nanopores [26][27] . However, nearly all biological membranes experience a potential difference of between 50 to 200 mV across them and molecular dynamics (MD) simulations of simple model nanopores show that application of a transmembrane electric field can hydrate an otherwise de-wetted hydrophobic constriction, which is thus rendered ion permeable [28][29] .…”